The MLL gene at chromosome band 11q23 is frequently rearranged in both acute myeloid and acute lymphoblastic leukemia. These translocations result in the formation of chimeric fusion proteins containing the N-terminus of MLL fused to the C-terminus of more than 70 different partner proteins. Although there are similarities in gene expression among the different MLL fusion proteins, MLL-AF4 (MA4), MLL-AF9 (MA9), and MLL-ELL (MEL) exhibit distinct gene expression profiles in leukemia cells obtained from patients. Many of the most common MLL partner proteins, including AF4, AF9, and ELL, are components of a super elongation complex (SEC) that is critical in transcriptional activation and elongation. Despite the identification that multiple MLL partner proteins are components of this complex, the basis for the differences in gene expression remains unclear. Many investigators refer to MLL-rearranged leukemia as a homogenous entity. However, the different fusions are found in different lineages. MA4 is almost exclusively found in pro-B ALL, MEL only in AML, and MA9 most commonly in AML but also in pre-B ALL. However, the basis for lineage specification by the different fusion partners is uncertain. Until now, it has not been possible to perform a direct comparison of the most common MLL fusions due to the lack of a tractable model of MLL-AF4 leukemia. We have developed a novel approach to express the MA4 fusion protein and have generated leukemia models using mouse and human hematopoietic stem and progenitor cells (HSPCs). Importantly, we have generated a faithful model of MA4 pro- B ALL. Using the unique reagents we have generated, we plan to examine the critical similarities and differences between these MLL fusion proteins. Each MLL fusion contains a triple FLAG tag that will permit the efficient purification of protein complexes and facilitate ChIP-seq to identify target genes. Although the partner protein complexes were identified several years ago, the complexes were immuno-precipitated using the partner proteins by themselves and not as MLL fusions. In addition, these purifications were performed in cell lines and not in primary leukemias induced by MLL fusions. In Aim 1, we will define the nature of the oncoprotein complexes formed in MLL-fusion transformed HSPCs and analyze their contribution to the initiation and maintenance of these leukemias. In Aim 2, we will identify critical downstream genes regulated by distinct MLL-fusion complexes in both myeloid and lymphoid cells. We will determine the genomic occupancy of each oncoprotein by ChIP-Seq. In this way we expect to identify those targets that are common to MLL and unique to each of the MLL-fusion proteins. In Aim 3, we will determine the cell of origin that is transformed in this MLL-fusion model system. We will express MA4, MEL, and MA9 in human stem and progenitor cells to determine whether each oncogene is able to induce leukemia in various progenitor cells and determine how cell of origin impacts leukemia type. Our studies are likely to have a large overall impact in the understanding of the mechanisms of transformation mediated by MLL fusion proteins and will provide key targets for therapeutic intervention.